Bottom Line:
Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix.Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding.RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

ABSTRACTPrevious physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144-180. Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

Figure 3: Identification of AKAP97 as RSP3. (A) Longitudinal view of the 96-nm repeat illustrating the relationship of the radial spokes to dynein structures (adapted from Porter and Sale 2000). DRC, dynein regulatory complex. (B) RII overlay of mutants defective in various components of the radial spoke. (C) Immunoblot of B with anti-RSP3.

Mentions:
To further localize AKAP97 within the radial spoke, we analyzed axonemes from several different radial spoke mutants by performing RII overlays. The results indicated that although AKAP97 is absent in pf14 (lacking the entire radial spoke), AKAP97 is present in pf17, which is defective for the radial spoke head only (Fig. 3a and Fig. B). This suggests that AKAP97 is associated with the radial spoke stalk. We also examined pf27, a radial spoke mutant in which five radial spoke components that are normally phosphorylated are underphosphorylated in the mutant (Piperno et al. 1981). Three of the five proteins, RSPs 2, 3, and 13, are present in greatly reduced amounts (Huang et al. 1981). The overlay revealed that AKAP97 is greatly reduced in pf27, and also appears to be shifted slightly more positive on an SDS-PAGE gel, consistent with an underphosphorylated state.

Figure 3: Identification of AKAP97 as RSP3. (A) Longitudinal view of the 96-nm repeat illustrating the relationship of the radial spokes to dynein structures (adapted from Porter and Sale 2000). DRC, dynein regulatory complex. (B) RII overlay of mutants defective in various components of the radial spoke. (C) Immunoblot of B with anti-RSP3.

Mentions:
To further localize AKAP97 within the radial spoke, we analyzed axonemes from several different radial spoke mutants by performing RII overlays. The results indicated that although AKAP97 is absent in pf14 (lacking the entire radial spoke), AKAP97 is present in pf17, which is defective for the radial spoke head only (Fig. 3a and Fig. B). This suggests that AKAP97 is associated with the radial spoke stalk. We also examined pf27, a radial spoke mutant in which five radial spoke components that are normally phosphorylated are underphosphorylated in the mutant (Piperno et al. 1981). Three of the five proteins, RSPs 2, 3, and 13, are present in greatly reduced amounts (Huang et al. 1981). The overlay revealed that AKAP97 is greatly reduced in pf27, and also appears to be shifted slightly more positive on an SDS-PAGE gel, consistent with an underphosphorylated state.

Bottom Line:
Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix.Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding.RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

ABSTRACTPrevious physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144-180. Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.